Synthesis and evaluation of thiazepines as interleukin-1beta converting enzyme (ICE) inhibitors

A series of monocyclic thiazepine inhibitors of interleukin-1beta converting enzyme (ICE) were synthesized in eight steps from commercially available intermediates. In vitro biological evaluation showed the thiazepines to be moderately potent ICE inhibitors, with the most active compound exhibiting an IC50 value of 30 nM in an enzyme inhibition assay. Compounds of this class possessed good selectivity against the related enzymes caspase-3 and caspase-8.     

Proteasome inhibitors and immunosuppressive drugs promote the cleavage of eIF4GI and eIF4GII by caspase-8-independent mechanisms in Jurkat T cell lines

Previously, we have shown that translation eukaryotic initiation factor (eIF) 4GI is cleaved during anti-Fas-mediated apoptosis. Here, we have investigated the effects of the proteasome inhibitors, MG132 and lactacystin, and the immunosuppressants, 2-amino-2[2-(4-octylphenyl)ethyl]-1,3,propane diol (FTY720) and cyclosporin A, on the integrity of eIF4GI and eIF4GII in T cells. Using wild-type Jurkat T cells, we show that the proteasome inhibitors MG132 and lactacystin promote the cleavage of eIF4G, activate caspase-8 and caspase-3-like activities and decrease cell viability. Furthermore, MG132 also promotes the cleavage of eIF4G and the activation of caspase-3-like activity in a caspase-8-deficient Jurkat cell line which is resistant to anti-Fas-mediated apoptosis. Using specific anti-peptide antisera, we show that both eIF4GI and eIF4GII are cleaved in either cell line in response to MG132 and lactacystin. In response to such treatments, we demonstrate that the fragments of eIF4GI generated include those previously observed with anti-Fas antiserum together with a novel product which lacks the ability to interact with eIF4E. In contrast, cells treated with the immunosuppressants FTY720 and cyclosporin A appear to contain only the novel cleavage fragment of eIF4GI and to lack those characteristic of cells treated with anti-Fas antiserum. These data suggest that caspase-8 activation is not required for apoptosis and eIF4G cleavage mediated by proteasome inhibitors and immunosuppressants in human T cells.     

Kinetic and structural characterization of caspase-3 and caspase-8 inhibition by a novel class of irreversible inhibitors

Because of their central role in programmed cell death, the caspases are attractive targets for developing new therapeutics against cancer and autoimmunity, myocardial infarction and ischemic damage, and neurodegenerative diseases. We chose to target caspase-3, an executioner caspase, and caspase-8, an initiator caspase, based on the vast amount of information linking their functions to diseases. Through a structure-based drug design approach, a number of novel β-strand peptidomimetic compounds were synthesized. Kinetic studies of caspase-3 and caspase-8 inhibition were carried out with these urazole ring-containing irreversible peptidomimetics and a known irreversible caspase inhibitor, Z-VAD-fmk. Using a stopped-flow fluorescence assay, we were able to determine individual kinetic parameters of caspase-3 and caspase-8 inhibition by these inhibitors. Z-VAD-fmk and the peptidomimetic inhibitors inhibit caspase-3 and caspase-8 via a three-step kinetic mechanism. Inhibition of both caspase-3 and caspase-8 by Z-VAD-fmk and of caspase-3 by the peptidomimetic inhibitors proceeds via two rapid equilibrium steps followed by a relatively fast inactivation step. However, caspase-8 inhibition by the peptidomimetics goes through a rapid equilibrium step, a slow-binding reversible step, and an extremely slow inactivation step. The crystal structures of inhibitor complexes of caspases-3 and -8 validate the design of the inhibitors by illustrating in detail how they mimic peptide substrates. One of the caspase-8 structures also shows binding at a secondary, allosteric site, providing a possible route to the development of noncovalent small molecule modulators of caspase activity.     

Activation of caspases is required for osteoblastic differentiation

Previous studies have shown that mouse osteoblastic MC3T3-E1 cells undergo apoptosis when exposed to a mixture of proinflammatory cytokines. Bone morphogenetic protein (BMP)s are important regulators of osteoblast differentiation. Because regulation of osteoblastic differentiation is poorly understood, we sought to determine if BMP-4-induced differentiation of osteoblastic cells depends on the activity of the key apoptotic proteases, i.e. the caspases. BMP-4 induced the growth arrest and differentiation of osteoblastic cell line MC3T3-E1, as evidenced by the appearance of osteoblastic phenotypes such as alkaline phosphatase (ALP) activation and parathyroid hormone (PTH)-dependent production of cAMP. Surprisingly, BMP-4 induced transient and potent activation of caspase-8, caspase-2, and caspase-3, in this order. However, no apoptosis or necrosis in BMP-4-treated cells could be detected by FACS using annexin-V/propodium iodine double staining. Peptide inhibition of caspase activity led to a dramatic reduction in ALP activation and PTH-induced production of cAMP in BMP-4-treated cells. Although BMP-4 treatment resulted in cell-cycle G0/G1 arrest as detected by FACS cell-cycle analysis, caspase inhibitors (caspase-8, caspase-2, and caspase-3 inhibitors) could block the G0/G1 arrest in MC3T3-E1 cells. Taken together, these results confirm a unique and unanticipated role for the caspase-mediated signal cascade in the differentiation of osteoblasts.     

p38-mediated regulation of an Fas-associated death domain protein-independent pathway leading to caspase-8 activation during TGFbeta-induced apoptosis in human Burkitt lymphoma B cells BL41

On binding to its receptor, transforming growth factor beta (TGFbeta) induces apoptosis in a variety of cells, including human B lymphocytes. We have previously reported that TGFbeta-mediated apoptosis is caspase-dependent and associated with activation of caspase-3. We show here that caspase-8 inhibitors strongly decrease TGFbeta-mediated apoptosis in BL41 Burkitt's lymphoma cells. These inhibitors act upstream of the mitochondria because they inhibited the loss of mitochondrial membrane potential observed in TGFbeta-treated cells. TGFbeta induced caspase-8 activation in these cells as shown by the cleavage of specific substrates, including Bid, and the appearance of cleaved fragments of caspase-8. Our data show that TGFbeta induces an apoptotic pathway involving sequential caspase-8 activation, loss of mitochondrial membrane potential, and caspase-9 and -3 activation. Caspase-8 activation was Fas-associated death domain protein (FADD)-independent because cells expressing a dominant negative mutant of FADD were still sensitive to TGFbeta-induced caspase-8 activation and apoptosis. This FADD-independent pathway of caspase-8 activation is regulated by p38. Indeed, TGFbeta-induced activation of p38 and two different inhibitors specific for this mitogen-activated protein kinase pathway (SB203580 and PD169316) prevented TGFbeta-mediated caspase-8 activation as well as the loss of mitochondrial membrane potential and apoptosis. Overall, our data show that p38 activation by TGFbeta induced an apoptotic pathway via FADD-independent activation of caspase-8.     

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis is dependent on activation of cysteine and serine proteases

We examined the role of caspases and serine protease(s) in cell death induced by tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). After incubation of adenocarcinoma cells with TRAIL, caspase-3, -8 were activated and the cleavage of Bid induced the release of cytochrome c, from the mitochondria to the cytosol. Tetrapeptide inhibitors of caspase-1, -2, -3, and -8 suppressed DNA fragmentation and attenuated the release of cytochrome c, whereas inhibitors of caspase-5 did not. Interestingly, the general serine protease(s) inhibitor 4-(2-aminoethyl)benzylsulfonyl fluoride (AEBSF) resulted in the arrest of apoptosis. However, the AEBSF did not prevent the release of mitochondrial cytochrome c during TRAIL-induced apoptosis. From these results, we postulate that serine protease(s) may be involved in post-mitochondrial apoptotic events, that lead to the activation of the initiator, caspase-9.     

Synthesis and evaluation of novel 1-(2-acylhydrazinocarbonyl)-cycloalkyl carboxamides as interleukin-1beta converting enzyme (ICE) inhibitors

Novel 1-(2-acylhydrazinocarbonyl)cycloalkyl carboxamides were designed as peptidomimetic inhibitors of interleukin-1beta converting enzyme (ICE). A short synthesis was developed and moderately potent ICE inhibitors were identified (IC(50) values <100 nM). Most of the synthesized examples were selective for ICE versus the related cysteine proteases caspase-3 and caspase-8, although several dual-acting inhibitors of ICE and caspase-8 were identified. Several of the more potent ICE inhibitors were also shown to inhibit IL-1beta production in a whole cell assay (IC(50) < 500 nM).     

Inhibition of histone deacetylase activity enhances Fas receptor-mediated apoptosis in leukemic lymphoblasts

We recently reported that butyrate, an inhibitor of histone deacetylases, is capable of inducing Fas-independent apoptosis in the acute lymphoblastic leukemia cell line CCRF-CEM. Here we demonstrate that butyrate enhances Fas-induced apoptosis in this cell line. The application of different histone deacetylase inhibitors revealed that tetra-acetylated histone H4 is associated with the amplifying effect of butyrate on Fas-induced cell death. FasL, Fas, FADD, RIP, caspase-8, caspase-3, Bid, FLIP(S+L), FLASH and FAP-1, proteins known to act within the Fas-apoptosis cascade, showed no changes in their expression levels in cells treated with butyrate compared with untreated cells. Analyses of Fas-oligomerization and Western blotting as well as enzyme activity assays of caspase-2, caspase-3 and caspase-8 suggest that butyrate enhances Fas-induced apoptosis downstream of Fas but upstream of caspase-8 activation. In immunoprecipitation experiments a 37 kD butyrate-regulated protein was detected which specifically interacts with caspase-8.     

Pseudomonas aeruginosa exotoxin A induces human mast cell apoptosis by a caspase-8 and -3-dependent mechanism

Mast cells play an important role in both allergy and innate immunity. Recently, we demonstrated an active interaction between human mast cells and Pseudomonas aeruginosa leading to the production of multiple cytokines. Here, we show that both primary cultured human cord blood-derived mast cells and the human mast cell line HMC-1 undergo apoptosis as determined by single-stranded DNA (ssDNA) formation after stimulation with P. aeruginosa exotoxin A (ETA), a major toxin produced by this bacterium. ETA-induced ssDNA formation was completely inhibited by Z-VAD (where Z is benzyloxycarbonyl), which blocks multiple caspases, suggesting a role for caspases in this process. Active caspase-3 formation in mast cells after an ETA challenge was detected by both Western blotting and flow cytometry analysis. ETA-induced caspase-3 activity in human mast cells was demonstrated by the detection of a characteristic 23 kDa product of D4-GDI (where GDI is guanine nucleotide dissociation inhibitor), an endogenous caspase-3 substrate. Interestingly, a specific caspase-8 inhibitor, Z-IETD-fmk (where fmk is fluoromethyl ketone), blocked ETA-induced cleavage of D4-GDI, but a caspase-9 inhibitor (Z-LEHD-fmk) did not. Treatment of mast cells with caspase-3 inhibitor Z-DEVD-fmk or caspase-8 inhibitor Z-IETD-fmk reduced the generation of ssDNA induced by ETA, suggesting a role for caspase-8 and -3 in ETA-induced mast cell apoptosis. Furthermore, treatment of mast cells with ETA induced decreases of the short form and a long form (p43) of Fas-associated death domain protein (FADD)-like interleukin-1beta-converting enzyme (FLICE) (caspase-8)-inhibitory proteins (FLIPs), which are endogenous caspase-8 inhibitors. Taken together, these results suggest that ETA-induced mast cell apoptosis involves down-regulation of antiapoptotic proteins, FLIPs, and activation of caspase-8 and -3 pathways.     

Hydroxylation at C4' or C6 is essential for apoptosis-inducing activity of flavanone through activation of the caspase-3 cascade and production of reactive oxygen species

Previous studies demonstrated that hydroxyl groups play important roles in the antioxidative activities of flavonoids; however, the importance of structurally related hydroxylation in their apoptosis-inducing activities is still undefined. In the present study, flavanone with hydroxylation at C4' and C6 had a significant cytotoxic effect in human leukemia HL-60 cells accompanied by the occurrence of DNA ladders, apoptotic bodies, and hypodiploid cells, characteristics of apoptosis. The replacement of a hydroxyl group (OH) by a methoxyl (OCH3) group at C4' or C6 attenuated the apoptotic effect in cells, and there was no significant cytotocity of flavanone or flavanone with OH or OCH3 in C7-treated HL-60 cells. Induction of enzyme activity of caspase-3 and -9, but not caspase-1 and -8, accompanied by release of cytocrome C from mitochondria to cytosol and the appearance of cleaved of PARP (85 kDa), D4-GDI (23 kDa), and caspase-3 (p17/p15) fragments, was identified in 4'-OH- or 6-OH- flavanone-treated HL-60 cells. Caspase-3 and -9 inhibitors Ac-DEVD-FMK and Ac-LEHD-FMK, but not caspase-1 and -8 inhibitors Ac-YVAD-FMK and Ac-LETD-FMK, attenuated 4'-OH- or 6-OH-flavanone-induced cell death. And, inhibition of capsase-9 activity by Ac-LEHD-FMK suppresses caspase-3 protein procession induced by 4'-OH- and 6-OH-flavanone, indicative of caspase-9 activation locating upstream of caspase-3. A decrease in the antiapoptotic protein Mcl-1 and increases in the pro-apoptotic proteins Bax and Bad were found in 4'-OH- or 6-OH-flavanone-treated HL-60 cells. Induction of endogenous ROS production was detected in 4'-OH- or 6-OH-flavanone-treated HL-60 cells by the DCHF-DA assay. Antioxidants such as N-acetylcysteine (NAC), catalase (CAT), superoxide dismutase (SOD), and allopurinol (ALL), but not pyrrolidine dithiocarbamate (PDTC) or diphenylene iodonium (DPI), significantly inhibited 4'-OH- or 6-OH-flavanone-induced ROS production, with blocking of the apoptosis induced by 4'-OH- or 6-OH-flavanone. The apoptosis-inducing activity of 4'-OH- or 6-OH-flavanone was also observed in another leukemia cell line (Jurkat), but was not found in mature monocytic cells (THP-1) and normal human polymorphonuclear neutrophils (PMNs). This suggests that hydroxylation at C4' or C6 is important to the apoptosis-inducing activities of flavanone through ROS production, and that activation of the caspase-3 cascade, downstream of caspase-9 activation, is involved.     

The three-dimensional structure of caspase-8: an initiator enzyme in apoptosis.

BACKGROUND: In the initial stages of Fas-mediated apoptosis the cysteine protease caspase-8 is recruited to the cell receptor as a zymogen (procaspase-8) and is incorporated into the death-signalling complex. Procaspase-8 is subsequently activated leading to a cascade of proteolytic events, one of them being the activation of caspase-3, and ultimately resulting in cell destruction. Variations in the substrate specificity of different caspases have been reported. RESULTS: We report here the crystal structure of a complex of the activated human caspase-8 (proteolytic domain) with the irreversible peptidic inhibitor Z-Glu-Val-Asp-dichloromethylketone at 2.8 A resolution. This is the first structure of a representative of the long prodomain initiator caspases and of the group III substrate specificity class. The overall protein architecture resembles the caspase-1 and caspase-3 folds, but shows distinct structural differences in regions forming the active site. In particular, differences observed in subsites S(3), S(4) and the loops involved in inhibitor interactions explain the preference of caspase-8 for substrates with the sequence (Leu/Val)-Glu-X-Asp. CONCLUSIONS: The structural differences could be correlated with the observed substrate specificities of caspase-1, caspase-3 and caspase-8, as determined from kinetic experiments. This information will help us to understand the role of the various caspases in the propagation of the apoptotic signal. The information gained from this investigation should be useful for the design of specific inhibitors.     

Mechanism of Siglec-8-induced human eosinophil apoptosis: role of caspases and mitochondrial injury

Sialic acid binding immunoglobulin like lectin (Siglec)-8 crosslinking with specific antibodies causes human eosinophil apoptosis. Mechanisms by which Siglec-8 crosslinking induces apoptosis are not known. Peripheral blood eosinophils were examined for caspase, mitochondria and reactive oxygen species (ROS) involvement after incubating the cells with anti-Siglec-8 crosslinking Abs or control Abs, in the presence or absence of selective inhibitors. Siglec-8 crosslinking induced rapid cleavage of caspase-3, caspase-8, and caspase-9 in eosinophils. Selective caspase-8 and/or caspase-9 inhibitors inhibited this apoptosis. Siglec-8 crosslinking on eosinophils increased dissipation of mitochondrial membrane potential upstream of caspase activation. Rotenone and antimycin, inhibitors of mitochondrial respiratory chain components, completely inhibited apoptosis. Additional experiments with an inhibitor of ROS, diphenyleneiodonium, demonstrated that ROS was also essential for Siglec-8-mediated apoptosis and preceded Siglec-8-mediated mitochondrial dissipation. These experiments show that Siglec-8-induced apoptosis occurs through the sequential production of ROS, followed by induction of mitochondrial injury and caspase cleavage.     

p38 MAPK and MSK1 mediate caspase-8 activation in manganese-induced mitochondria-dependent cell death

Heavy metals are important regulators of cell apoptosis. Manganese (Mn(2+)) is a potent inducer of apoptosis in different cell types, but the precise mechanisms that mediate such effects are not well defined. We previously reported that Mn(2+) was a potent apoptotic agent in human B cells, including lymphoma B cell lines. We show here that Mn(2+)-induced cell death in human B cells is associated with caspase-8-dependent mitochondrial activation leading to caspase-3 activity and apoptosis. We used specific caspase-8 interfering shRNAs to reduce caspase-8 expression, and this also reduced Mn(2+)-induced caspase-3 activation and apoptosis. Mn(2+)-triggered caspase-8 activation is associated with a specific pathway, which is independent of Fas-associated death domain protein, and dependent on the sequential activation of p38-mitogen-activated protein kinase (p38 MAPK) and mitogen- and stress-response kinase 1 (MSK1). Inhibition of p38 activity using either pharmacological inhibitors or dominant-negative mutant forms of p38 blocked Mn(2+)-mediated phosphorylation of MSK1 and blocked subsequent caspase-8 activation. However, specific inhibitors and the expression of a dominant-interfering mutant of MSK1 only inhibited caspase-8 activation, but not p38 activity. These findings suggest a novel model for the regulation of caspase-8 during Mn(2+)-induced apoptosis based on the sequential activation of p38 MAPK, MSK1, caspase-8 and mitochondria, respectively.     

Arsenic trioxide sensitizes promonocytic leukemia cells to TNFalpha-induced apoptosis via p38-MAPK-regulated activation of both receptor-mediated and mitochondrial pathways

Treatment with the anti-leukemic drug arsenic trioxide (As(2)O(3), 1-4 microM) sensitizes U937 promonocytes and other human myeloid leukemia cell lines (HL60, NB4) to apoptosis induction by TNFalpha. As(2)O(3) plus TNFalpha increases TNF receptor type 1 (TNF-R1) expression, decreases c-FLIP(L) expression, and causes caspase-8 and Bid activation, and apoptosis is reduced by anti-TNF-R1 neutralizing antibody and caspase-8 inhibitor. The treatment also causes Bax translocation to mitochondria, cytochrome c and Omi/HtrA2 release from mitochondria, XIAP down-regulation, and caspase-9 and caspase-3 activation. Bcl-2 over-expression inhibits cytochrome c release and apoptosis, and also prevents c-FLIP(L) down-regulation and caspase-8 activation, but not TNF-R1 over-expression. As(2)O(3) does not affect Akt phosphorylation/activation or intracellular GSH content, nor prevents the TNFalpha-provoked stimulation of p65-NF-kappaB translocation to the nucleus and the increase in NF-kappaB binding activity. Treatments with TNFalpha alone or with As(2)O(3) plus TNFalpha cause TNF-R1-mediated p38-MAPK phosphorylation/activation. P38-MAPK-specific inhibitors attenuate the As(2)O(3) plus TNFalpha-provoked activation of caspase-8/Bid, Bax translocation, cytochrome c release, and apoptosis induction. In conclusion, the sensitization by As(2)O(3) to TNFalpha-induced apoptosis in promonocytic leukemia cells is an Akt/NF-kappaB-independent, p38-MAPK-regulated process, which involves the interplay of both the receptor-mediated and mitochondrial executioner pathways.     

Reconstitution of caspase-8 sensitizes JB6 cells to TRAIL

TRAIL induces apoptosis in various tumor cells. We report here that caspase-8 is required in TRAIL-induced cell death. Western blot analyses and enzyme assays showed that exposing Jurkat cells to TRAIL resulted in activation of caspases-8 followed by caspase-3 and -9. Acetyl-IETD-fluoromethylketone, a caspase-8 inhibitor, potently suppressed TRAIL-induced cell death compared to acetyl-DEVD-fluoromethylketone and acetyl-LEHD-fluoromethylketone, inhibitors of caspase-3 and caspase-9, respectively. JB6 cells, a caspase-8-deficient Jurkat variant, were completely resistant to TRAIL. However, reconstitution with a caspase-8, but not with caspase-2 or -3, sensitized JB6 cells to subsequent exposure to TRAIL. These results are indicative of the crucial function of caspase-8 in TRAIL-induced apoptosis in Jurkat cells.     

Synthesis and evaluation of novel dipeptidyl benzoyloxymethyl ketones as caspase inhibitors

We describe novel peptide-based caspase inhibitors. Potent and comparatively selective compounds containing a dipeptide scaffold and a substituted oxymethyl ketone as a warhead were developed. The newly synthesized compounds were tested for inhibition in in vitro enzymatic assays of caspases-1, -3, -6, -8, and -9. The benzyloxycarbonyl-phenylglycyl-aspartyl benzoyloxymethyl ketone (Z-Phg-Asp-CH2OCO-Ph, coded as HU44) was the most potent inhibitor of caspase-1 and caspase-3. Of several analogs of HU44 that were made, the beta-Asp methyl ester (2) is an effective inhibitor against caspase-3 and caspase-8, and less effective against caspase-1. These compounds did not inhibit caspase-6 and caspase-9 significantly.     

Caspase-3 feeds back on caspase-8, Bid and XIAP in type I Fas signaling in primary mouse hepatocytes

The TNF-R1 like receptor Fas is highly expressed on the plasma membrane of hepatocytes and plays an essential role in liver homeostasis. We recently showed that in collagen-cultured primary mouse hepatocytes, Fas stimulation triggers apoptosis via the so-called type I extrinsic signaling pathway. Central to this pathway is the direct caspase-8-mediated cleavage and activation of caspase-3 as compared to the type II pathway which first requires caspase-8-mediated Bid cleavage to trigger mitochondrial cytochrome c release for caspase-3 activation. Mathematical modeling can be used to understand complex signaling systems such as crosstalks and feedback or feedforward loops. A previously published model predicted a positive feedback loop between active caspases-3 and -8 in both type I and type II FasL signaling in lymphocytes and Hela cells, respectively. Here we experimentally tested this hypothesis in our hepatocytic type I Fas signaling pathway by using wild-type and XIAP-deficient primary hepatocytes and two recently characterized, selective caspase-3/-7 inhibitors (AB06 and AB13). Caspase-3/-7 activity assays and quantitative western blotting confirmed that fully processed, active p17 caspase-3 feeds back on caspase-8 by cleaving its partially processed p43 form into the fully processed p18 species. Our data do not discriminate if p18 positively or negatively influences FasL-induced apoptosis or is responsible for non-apoptotic aspects of FasL signaling. However, we found that caspase-3 also feeds back on Bid and degrades its own inhibitor XIAP, both events that may enhance caspase-3 activity and apoptosis. Thus, potent, selective caspase-3 inhibitors are useful tools to understand complex signaling circuitries in apoptosis.     

Development of cell death-based method for the selectivity screening of caspase-1 inhibitors

Caspase-1 selective inhibitors are novel therapeutic agents for inflammatory diseases. Selectivity assays for caspases can be initiated with purified enzyme, making these assays very costly and time consuming. Therefore, there is a need to develop a fast and reliable cell-based assay, which can be used for the selectivity screening of multiple caspases in a biologically relevant context in a single assay. In this study, we have developed an assay in which DNA fragmentation, a hallmark of apoptosis, of Jurkat cell line was examined post induction with etoposide in the presence or absence of inhibitors of caspases 1, 3, 8, 9 and pan-caspase inhibitors. We observed that caspases-3, -8, -9 and pan caspase inhibitors resulted in significant inhibition of etoposide-induced DNA fragmentation. However, caspase-1 specific inhibitor failed to prevent DNA fragmentation, suggesting that either caspases belonging to caspase-1 family (1, 4 and 5) are not present in the Jurkat cells or might not be involved in the etoposide-induced DNA fragmentation. Since the inhibition of caspases 3, 8 and 9 is accompanied by the down regulation of the activity of a cascade of caspases (caspases 2, 6, 7, 9 and 10), selectivity of caspase-I inhibitors can be ascertained for the above panel (caspases 2, 6, 7, 8, 9 and 10) of caspases from this single assay.     

Solid phase synthesis of selective caspase-3 peptide inhibitors

A robust method for the solid phase synthesis of a series of selective caspase-3 peptide inhibitors is described. The inhibitors can be obtained after cleavage from the solid support without further purification.